Summary of work: The mitochondrial DNA (mtDNA) accumulates high levels of oxidative damage owing to its proximity to the electron transport chain, where most reactive oxygen species are generated. Oxidative damage can cause mutations, deletions and lead to cell death. 8-hydroxyguanine, a major oxidative DNA lesion, accumulates with age in the mtDNA. Repair of oxidative DNA damage is carried out by the base excision repair (BER) system. We investigate the repair mechanisms for the removal of DNA lesions, particularly oxidized bases, from the mitochondrial genome. We have investigated the role of the oxoguanine DNA glycosylase 1 (Ogg1) in mtDNA repair in mice that are defective in this enzyme. Our results suggest that Ogg1 plays a crucial role in the repair of oxidative damage in mitochondria and is probably the only 8OHdG glycosylase in these organelles. In human cells two distinct Ogg1 isoforms are expressed, alpha and beta. Beta-Ogg1 localizes only to mitochondria and was believed to provide the 8-OHdG glcycosylase activity. We purified recombinant b-Ogg1 and found that the protein lacks glycosylase activity. Site-directed mutagenesis studies identified two aminoacids that are found in the b-isoform that render the a-isoform inactive. We also found that approximately 10% of a-Ogg1 localizes to mitochondria and may provide the 8-OHdG glycosylase activity. NTH is the other major glycosylase for repair of oxidative DNA damage. We investigated DNA repair in liver mitochondria from mice deficient in this enzyme. We found that those mitochondrial extracts can not repair thymine glycol lesions in DNA, indicating that the NTH enzyme is responsible for the repair of these lesions in DNA. We observed some residual incision acitvity for other oxidized pyrimidines in extracts from NTH knockou mice, suggesting that some minor backup pathways my exist in mitochondria for the removal of these lesions. These DNA glycosylases are encoded in the nucleus and transported to mitochondria; however there is very limited information on the regulation of mitochondrial BER. We measured BER activities in mitochondria that lack mtDNA (rho-). Despite the absence of mtDNA a complete mitochondrial BER was present, and most activities were only slightly decreased compared to wt mitochondria. Interestingly, nuclear BER activities were also affected by the absence of mtDNA, suggesting an interesting cross-talk between BER in both compartments. We are now investigating whether mammalian mitochondria have other repair pathways that operate in the nucleus, such as mismatch repair. The p53 protein has recently been associated with BER in the nucleus. We investigated whether p53 participates in BER in mitochondria and found that mitochondrial extracts from p53 null mouse liver have normal levels of DNA glycosylase and AP-endonulcease activities. DNA repair synthesis incorporation is slightly decrease but can be stimulated by addition of recombinant p53. DNA polymerase gamma activity, measured in a gap-filling assay, was also decreased in extracts from the knockout mice. Our results suggest that p53 participates in the nucleotide incorporation step in mitochondrial BER. Calorie restriction (CR) is the only intervention known so far that slows aging. We studied DNA repair activities in mitochondriaand nuclei from caloric restricted mice to determine whether DNA repair is affected by such dietary changes. We found that CR modulates nuclear and mitochondrial BER differentially. Nuclear BER is significantly up-regulated in CR animals while mitochondria BER is only slightly higher. We also observed organ specific differences in the response to CR. Our results indicate that a general up-regulation of BER does not occur during CR. One strenght of our studies is that we assay for DNA repair activity and measure the actual occurrence of the lesions in DNA using HPLC and other analytical techniques.